Recent evidence in the field suggests that Cdk6 is an early target of pluripotency, suggesting it both promotes self-renewal and blocks differentiation, both characteristics of cancer stem cells. Recent work from our lab has shown that Cdk6 binds Eya2 and that the expression of Cdk6 results in degradation of the Eya2 protein (Kohrt et al, 2014). Eya2 is a member of the Eya family of proteins that are pro-proliferative, important in development, and misregulated in several types of cancer. The objective of experiments outlined in this proposal is to investigate the functional significance of the interaction of Cdk6 and Eya2. The Cdk6-promoted degradation of the Eya2 protein suggests that Cdk6 may regulate Eya2 activity, a function that could be important in both development and in cancer. The proposed experiments are designed to test the innovative hypothesis that inactive Cdk6 enzyme (Cdk6 molecules present in late M or early G1 phase) constrain cell division by promoting the degradation of the pro-mitotic Eya2 molecule. These approaches are aimed at discovering the mechanism of Cdk6-mediated degradation of the Eya2 protein, the functional outcomes of complex formation and the impact of Eya2 degradation on cell division. The first aim will examine the influence of cell cycle phase, sub-cellular localization, and components of the cell cycle machinery on Cdk6-Eya2 complex formation, and on Cdk6-mediated degradation of Eya2. This aim will also determine if Eya2 degradation influences cell division. The second aim will investigate the specificity of the interaction of Cdk6 with Eya2. It will seek to determin what domains of each protein are required for complex formation and if other cdks bind Eya2 or if other Eya family members bind Cdk6. The third aim will determine what elements of the degradation machinery are involved in Cdk6-mediated degradation of Eya2, both by looking at likely candidate proteins and by an unbiased biochemical approach. This proposal meets the stated goals of the AREA grant by 1) supporting meritorious research at an undergraduate institution, 2) creating opportunities for scientists (PI, technician, undergraduates) to participae in NIH programs, 3) strengthening the research environment of Connecticut College and 4) exposing undergraduate students to meritorious research. Of the undergraduate students who have studied in the principal investigator's lab, 33% have completed or are enrolled in Ph.D. programs, another 33% have gone on to earn medical, veterinary, dental or nurse practitioner degrees while 25% are pursuing careers in science or teaching. These outcomes indicate that research at undergraduate institutions inspires future scientists and health professionals.